Hematopoietic/lymphoid cell differentiation is an area of intense investigation, and a number of cytokines and other factors have been identified which modulate growth, differentiation, and functional activity of hematopoietic and lymphoid cells. These include a number of microbial products such as LPS, TSST-1, erythrogenic toxins A and C, and several Mycoplasma products. In the process of examining tumor cell line conditioned media for cytokines and factors with differentiation activity, several years ago we identified, purified, and began characterizing a 48 kd factor termed P48. The biochemical characteristics and biologic activities of this factor include induction of monocytic differentiation and cytolytic activity in the HL-60 (human promyelocytic) cell line, growth inhibition (but not nonspecific toxicity) toward a large panel of other human and mouse cell lines, induction of secretion of IL-1 and TNF-alpha by human peripheral blood monocytes, and induction of accumulation of mRNAs for IL1- alpha, IL1-beta, lL6, and TNF-alpha in monocytes and monocyte-like cell lines. Over the past three years, we have further characterized this factor including cDNA and genomic cloning, sequencing, expression of the gene in E. coli, and purification of the biologically active recombinant protein. We have found that P48 represents a Mycoplasma fermentans gene product, and sequence analysis revealed a number of characteristics of a lipoprotein suggesting that P48 may be functionally similar to bacterial LPS. This revised grant proposal is designed to further characterize the biochemistry, molecular biology, and biology of P48 in detail including (I) characterization of the surface or cytoplasmic receptor for P48 on monocytes and monocyte-like cell lines and possible involvement of serum binding proteins; (2) examination of the biochemistry and functional significance of post-translational modification (e.g. lipid and/or carbohydrate) of the molecule; (3) examination of the mechanism of P48 induction of inflammatory cytokines with comparison to LPS; and (4) expression in eukaryotic systems including Baculovirus/insect cells and CHO cells P48 is an important immunomodulatory molecule which may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian hematopoietic cell differentiation and cytokine biosynthesis.